Sestrin2 protects rat cardiomyocytes from hypoxia-reoxygenation injury through Nrf2/HO-1 signaling pathway

Abstract

Objective To investigate the role and underlying mechanism of Sestrin2 in hypoxia/reoxygenation injury of rat cardiomyocytes. Methods H9C2 cardiomyocytes were cultured in vitro and inflicted to establish hypoxia/reoxygenation injury (H/R) model. Thus, the cells were divided into control group, hypoxia 4-hour group (H4h group), and hypoxia 4-hour + reoxygenation 2- and 4-hour groups (H4hR2h and H4hR4h groups). Cell survival rate was measured by cell counting kit-8 (CCK8) assay; Annexin V/7AAD staining with flow cytometry was used to detect cell apoptosis. Micro-enzymatic method was used to detect the content of myocardial injury marker, lactate dehydrogenase (LDH). Then H9C2 cardiomyocytes were divided into normal control group, hypoxia 4-hour+si-NC group (negative control), hypoxia 4-hour+Sestrin2 siRNA group, hypoxia/reoxygenation 4 hour+si-NC group and hypoxia/reoxygenation 4 h+Sestrin2 siRNA group. Recombinant siRNA for Sestrin2 silencing and negative control (si-NC) were constructed and transfected into H9C2 cardiomyocytes with liposomes. The expression of Sestrin2, Nrf2 and HO-1 proteins was detected by Western blotting. Flow cytometry was used to measure the cell apoptosis. Results The cell viability of cardiomyocytes was significantly lower (P < 0.05), and the apoptosis (P < 0.05) as well as LDH level (P < 0.01) was obviously increased in the H4hR4h group than the control group. The expression of Sestrin2 protein was increased in both hypoxia and reoxygenation groups when compared with the control group (P < 0.01), with the level in the H/R groups higher than that in the H groups (P < 0.01). Transfected of Sestrin2 siRNA significantly knocked down the protein expression (P < 0.05), enhanced the cell apoptosis (P < 0.05), and decreased the expression levels of Nrf2 (P < 0.01) and HO-1 (P < 0.05). Conclusion Sestrin2 exerts protective effect on oxidative damage in H9C2 cardiomyocytes induced by H/R injury, which might be due to its activating Nrf2/HO-1 related signaling pathway.

Keywords

• sestrin 2
• myocardial hypoxia-reoxygenation injury
• apoptosis